Attenuating Mutations in nsP1 Reveal Tissue-Specific Mechanisms for Control of Ross River Virus Infection

Ross River virus (RRV) is one of a group of mosquito-transmitted alphaviruses that cause debilitating, and often chronic, musculoskeletal disease in humans. Previously, we reported that replacement of the nonstructural protein 1 (nsP1) gene of the mouse-virulent RRV strain T48 with that from the mouse-avirulent strain DC5692 generated a virus that was attenuated in a mouse model of disease. Here we find that the six nsP1 nonsynonymous nucleotide differences between strains T48 and DC5692 are determinants of RRV virulence, and we identify two nonsynonymous nucleotide changes as sufficient for the attenuated phenotype. RRV T48 carrying the six nonsynonymous DC5692 nucleotide differences (RRV-T48-nsP1(6M)) was attenuated in both wild-type and Rag1(-/-) mice. Despite the attenuated phenotype, RRV T48 and RRV-T48-nsP1(6M) loads in tissues of wild-type and Rag1(-/-) mice were indistinguishable from 1 to 3 days postinoculation. RRV-T48-nsP1(6M) loads in skeletal muscle tissue, but not in other tissues, decreased dramatically by 5 days postinoculation in both wild-type and Rag1(-/-) mice, suggesting that the RRV-T48-nsP1(6M) mutant is more sensitive to innate antiviral effectors than RRV-T48 in a tissue-specific manner. In vitro, we found that the attenuating mutations in nsP1 conferred enhanced sensitivity to type I interferon. In agreement with these findings, RRV T48 and RRV-T48-nsP1(6M) loads were similar in mice deficient in the type I interferon receptor. Our findings suggest that the type I IFN response controls RRV infection in a tissue-specific manner and that specific amino acid changes in nsP1 are determinants of RRV virulence by regulating the sensitivity of RRV to interferon.